Cryopreservation of Zebrafish Germ Cells by Vitrifying Gonads

Abstract

The zebrafish is an indispensable model system for biomedical research, including human diseases. As the number of mutant and transgenic zebrafish lines exponentially grows, so does the cost of labor and space to maintain these models. Successful cryopreservation of zebrafish gametes and embryos would significantly reduce such maintenance costs, avoid risks of infection and genetic drift associated with continuous breeding, and serve as a major steppingstone towards the conservation of other fish species under threat of extinction. While cryopreservation of fish sperm is somewhat successful, fish eggs and embryos remain challenging to cryopreserve due to several obstacles, such as their susceptibility to the toxicity of cryoprotective agents (CPAs), chilling injury, and intracellular ice formation. Recent studies demonstrated that fish germ cells isolated from testes or ovaries can colonize gonads upon transplantation into recipients and give rise to fertile offspring. Hence, the aim of this study was to develop a reliable cryopreservation method for zebrafish germ cells by vitrifying gonads. Vitrification is an ice-free cryopreservation method requiring the presence of high concentrations of CPAs that are toxic to cells. To minimize CPA toxicity, Dr. Eroglu’s lab developed a novel proprietary vitrification medium that was used in the present study. A series of experiments were carried out using both male and female zebrafish gonads, and the viability of germ cells were assessed after exposure to conventional and minimally toxic proprietary vitrification media, as well as after vitrification with the latter. The CPA exposure experiments revealed that the treatment of both male and female gonads with a conventional vitrification medium significantly reduced cell viability compared to untreated controls (58-66% vs. 86-95%, respectively). In contrast, the same exposure steps to the proprietary vitrification medium did not induce any significant toxicity resulting in viability rates similar to those of controls (95-97%). Importantly, a vast majority of gonad cells (88-93%) remained viable after vitrification of zebrafish gonads using the proprietary vitrification medium. Taken together, these findings suggest that cryopreservation of zebrafish germ cells is feasible using the proprietary vitrification medium.